Campylobacter jejuni (C. jejuni), a gram-negative microaerophilic bacterium, is a leading cause of bacterial diarrhea and enterocolitis in children and adults in both developing and developed countries (Walker R I et al, Microbiol. Rev. 50 (1): 81-94, 1986; Kim N W et al, J. Bacteriol. 174 (11):3494-3498, 1992; Chan V L and Bingham H L, Gene 101:51-58, 1991). Clinical symptoms of Campylobacter infections range from watery diarrhea to inflammatory dysentery and bloody diarrhea (Cover TL and Blaser N J, Ann. Rev. Ned. 40:269-285, 1989; Walker R I et al, supra). Complications from C. jejuni infections have included Guillain-Barre syndrome, a neurological disease which may lead to respiratory paralysis and death, toxic megacolon, acute mesenteric adenitis syndrome, and reactive arthritis (Kaldor J and Speed B R, British Medical J. 288:1867-1870, 1984; Johnson K et al, Acta. Med. Scand. 214:165-168, 1983; Walker R I et al, supra).
Campylobacter jejuni is commonly found in surface water, in animals such as cattle, sheep, goats, swine and poultry, in industrial wastes, and in many different types of foods including unpasteurized dairy products. Human pets such as dogs, cats and birds may also be infected with C. jejuni and may transmit the bacterium to humans. (Cover T L and Blaser M J, Ann. Rev. Med. 40:269-285, 1989; and Penner, J. L., Clin. Micro. Rev. 1:157-172, 1988).
A number of different strategies have been developed for detecting and identifying C. jejuni in food samples, water, and environmental and clinical specimens. C. jejuni has been differentiated from other pathogenic camplylobacteria such as C. coli and C. lari, by its ability to hydrolyze hippurate (Cowan S T, Int. Bull. Bact. Nom. Tax. 5:97, 1955). The enzyme benzoyl-glycine aminohydrolase, commonly known as hippuricase, is responsible for cleaving N-benzoylglycine (hippurate) into benzoic acid and glycine. The enzyme is present in C. jejuni and it is either absent or non-functional in other campylobacteria, including C. coli and C. lari.
Hippuricase activity has been shown in a variety of microorganisms including Actinobacillus, Aerobacter aerogenes, Aerococcus viridans, Campylobacter jejuni, certain Enterobacteriaceae, some species in the genera Bacillus, Benekea, Corynebacterium, Listeria, Pediococcus, Pseudomonas, Streptococcus, Mycobacterium, and Nocardia, and in the fungi Fusarium semiticum, and Streptomyces. Hippuricase from Streptococcus (Braunstein H et al., Am. J. Clin. Pathol. 51:207, 1969; Facklam R R et al., Appl. Microbiol. 27:107, 1974), Pseudomonas (Kameda et al., Chem. Pharm. Bull. Tokyo 16:1023, 1968), and Fusarium semicticum (Rohr M., Monatshefte fur Chemie 99:2255-2277, 1968), has been partially characterized. The molecular weight has not been determined but it is estimated to be between 70,000 to 100,000. The protein has been shown to be antigenic and antisera has been prepared to the streptococcal enzyme. Only fragmentary evidence on hippuricase is available for other organisms. It has been postulated that hippuricase is used by microorganisms as a mechanism of generating either an amino acid or benzoic acid as a substrate for metabolism.
Methods currently used to detect hippuricase activity in Campylobacter jejuni include the standard procedure for streptococci by Hwang and Ederer (Hwang M and Ederer G M, J. Clin. Microbiol. 1:114, 1975; Edberg S C and Samuels S, J. Clin. Microbiol. 3:49, 1976) based on the detection of glycine using a ninhydrin reagent. Hippuricase activity has also been detected through the determination of the second product, benzoic acid (Ottow J C G, J. Appl. Bacteriol. 37:15, 1974; Ayers and Rupp, supra, 1922).
These methods are not adequate to allow accurate detection of C. jejuni. For example, C. coli may yield weak hippuricase reactions, and C. jejuni may give weak or no hippuricase activity (Totten P A et al., J. Clin. Microbiol. 25:1747-1752, 1987). The currently used methods for detecting hippuricase activity are also cumbersome due to the need to culture the organisms prior to testing. Further, nonconventional means such as gas-liquid chromatography must be used to quantitate the hydrolysed benzoic acid product in very weak reactions (Bar W and Fricke G, J. Clin. Microbiol. 25:1776-1778, 1987; Wallace P L et al, J. Clin. Microbiol. 25:3766-1768, 1987).
DNA probes have been developed for detection and identification of C. jejuni and other campylobacteria. None of the probes have been fully characterized and the nature of the gene products and their functions are unknown. Romaniuk and Trust (Mol. Cell. Probes 3:133-142, 1989) used partial rRNA sequence information of campylobacteria to develop oligonucleotide probes to 16 S ribosomal RNA. One of these three oligo probes is reported to specifically identify C. jejuni and C. lari, and the other two are reported to be specific for C. jejuni, C. coli, and C. lari. Barns et al (European Patent Application No. 89306594.6, published on Jan. 10, 1990 as No. 0,350,205) disclose small nucleic acid probes which are reported to be capable of specifically hybridizing to ribosomal RNA of C. jejuni, C. coli and C. laridis and not to rRNA or rRNA genes of Pseudomonas aeroginosa, E. coli, or Salmonella typhimurium.
Taylor and Hiratsuka (Mol. Cell. Probes 4:261-271, 1989) developed two DNA probes using cloned C. jejuni genomic fragments obtained by screening a lambda gtll library with an antiserum prepared against a 46 kD major outer membrane protein of C. jejuni One of the probes (pDT1728) is reported to be C. jejuni specific, while the other (pDT1719) is reported to detect C. jejuni and C. coli but has lower sensitivity for the latter. Rashtchian (U.S. Pat. No. 4,785,086) describes a DNA probe capable of hybridizing to DNA of at least 80% of C. jejuni bacteria, as well as DNA in other campylobacteria.
Blaser et al. (U.S. Pat. No. 5,200,344) disclose antigenic compositions, and antibodies against the antigenic compositions, for use in diagnostic testing for C. jejuni or C. coli. The antigens in the compositions are obtained by acid extraction of surface antigens of C. jejuni and/or C. coli. The antigenic compositions described by Blaser et al. are not capable of differentiating C. jejuni and C. coli.
Antibody probes developed for campylobacteria are limited to the serotype antisera used to establish the epidemiological relationship between various Campylobacter isolates. These antibody probes are used only after the isolates have been identified as belonging to the genus by using the conventional methods of biochemical reactions, morphology, cultural and Gram reactions. The serotyping systems of Penner and Lior utilize antibody reactions against heat stable and heat-labile antigens respectively (Penner J L, Clin. Micro. Rev. 1:157-172, 1988; and Lior H et al., J. Clin. Microbiol. 15:761-768, 1982). The thermostable antigens have been proven to be the lipopolysaccharide antigens located in the outer membrane of the organism, and are detected through passive hemagglutination of erythrocytes. The Lior system uses antisera that have been absorbed with thermostable antigens of the homologous serostrain, and detects thermolabile antigens by means of slide agglutination of cell suspensions. The mixture of heat-labile antigens is as yet uncharacterized.